The mammalian intestinal epithelium is a dynamic system in which proliferation, migration, differentiation, and apoptosis are carefully orchestrated throughout the lifespan of an organism. Proliferation of multipotent stem cells and progenitor (also called transient amplifying) cells is confined to the crypt epithelium of the intestine. Recent studies have identified several important signaling pathways that are involved in maintaining intestinal epithelial cell homeostasis. Among these, the Wnt pathway is critical for the proliferation and self-renewal of multipotent stem cells, as demonstrated by the marking of these cells by Wnt targets such as LGR5, OLFM4 and ASCL2. However, the exact intracellular mediators of proliferation of intestinal stem cells and progenitor cells have not been definitively identified. Further characterization of the molecular network that controls crypt cell proliferation will increase the understanding of the precise mechanism that regulates intestinal epithelial cell proliferation and help elucidate the molecular pathogenesis underlying certain disease processes such as inflammatory bowel disease and neoplasm of the intestinal tract. The long term goal of this research project is to understand the molecular mechanisms regulating intestinal epithelial cell proliferation. The project supported by the grant (DK052230) on which this renewal application is based established that the zinc finger transcription factor, Kr[unreadable]ppel-like factor 5 (KLF5), plays an important role in regulating proliferation of intestinal epithelial cells. Expression of KLF5 is highly enriched in the proliferating crypt epithelial cell compartment of the intestinal epithelium. In vitro, KLF5 exhibits a pro-proliferative effect on intestinal epithelial cells. In addition, we showed that KLF5 is a mediator for the proliferative or regenerative response of intestinal epithelial cells to external stressors such as lipopolysaccharide (LPS), pathogenic bacterial infection by Citrobacter rodentium, and dextran sodium sulfate (DSS)-induced colitis. Furthermore, we demonstrated that KLF5 is a crucial mediator for formation of intestinal adenomas in mice with ApcMin mutation or combined ApcMin and activating KRAS mutations. Based on these observations, we propose the central hypothesis that KLF5 is an essential intracellular regulator of proliferation of intestinal crypt stem cells and progenitor cells. We propose three specific aims to test this hypothesis: (1) To investigate the effect of conditional deletion of Klf5 from the intestinal epithelium on intestinal epithelial cell proliferation in vivo;(2) To establish an essential role for KLF5 in mediating the oncogenic effect of activated KRAS in intestinal epithelial cells in vivo;and (3) To investigate the mechanism by which the Wnt pathway regulates KLF5 protein stability. These experiments will provide definitive evidence that KLF5 is an essential in vivo mediator controlling proliferation of intestinal epithelial cells. In addition, they will also establish that KLF5 mediates the oncogenic activity of mutated KRAS and activated Wnt signaling. The results may demonstrate that KLF5 is a potential therapeutic target for diseases with increased proliferation such as colon cancer. PUBLIC HEALTH RELEVANCE: Diseases of the gastrointestinal (GI) tract represent a significant health care burden. They are often caused by perturbation or imbalance in intestinal epithelial cell homeostasis. Investigating the mechanisms that regulate proliferation of intestinal epithelial cells at a molecular and cellular level will help understand the pathophysiology of many GI diseases such as inflammatory bowel disease and cancers of the GI tract, including colorectal cancer. The project stated herein will help gain new knowledge on how proliferation of intestinal epithelial cells is regulated and may help establish potentially novel therapeutic approaches to GI diseases.